1. Cross-Reference
U.S. patent application Ser. No. 10/076,042 entitled METHOD AND APPARATUS FOR SAMPLE INJECTION ON MICROFABRICATED DEVICES, concurrently filed on Feb. 11, 2002, which is assigned to MicroChem Solutions, Inc., the assignee of the present invention, and which is fully incorporated by reference herein.
2. Field of the Invention
The present invention relates generally to miniature instrumentation for conducting chemical reaction and/or bio-separation, and diagnostics and/or analysis related thereto, and more particularly, to the introduction of samples to the chemical reaction and/or bio-separation channels in microfabricated devices.
3. Description of Related Art
Bioanalysis, such as DNA analysis, is rapidly making the transition from a purely scientific quest for accuracy to a routine procedure with increased, proven dependability. Medical researchers, pharmacologists, and forensic investigators all use DNA analysis in the pursuit of their tasks. Yet due to the complexity of the equipment that detects and measures DNA samples and the difficulty in preparing the samples, the existing DNA analysis procedures are often time-consuming and expensive. It is therefore desirable to reduce the size, number of parts, and cost of equipment, to make easy sample handling during the process.
One type of DNA analysis instruments separates DNA molecules by relying on electrophoresis. Electrophoresis techniques could be used to separate fragments of DNA for genotyping applications, including human identity testing, expression analysis, pathogen detection, mutation detection, and pharmacogenetics studies. The term electrophoresis refers to the movement of a charged molecule under the influence of an electric field. Electrophoresis can be used to separate molecules of different electrophoretic mobilities in a given separation medium. DNA fragments are one example of such molecules.
There are a variety of commercially available instruments applying electrophoresis to analyze DNA samples. One such type is a multi-lane slab gel electrophoresis instrument, which as the name suggests, uses a slab of gel on which DNA samples are placed. Electric charges are applied across the gel slab, which cause the DNA sample to be separated into DNA fragments of different masses.
Another type of electrophoresis instruments is the capillary electrophoresis instrument. Capillary electrophoresis can be considered as one of the latest and most rapidly expanding techniques in analytical chemistry. Capillary electrophoresis refers to a family of related analytical techniques that uses very strong electric fields to separate molecules within narrow-bore capillaries (typically 20-100 um internal diameter). Capillary electrophoresis techniques are employed in seemingly limitless applications in both industry and academia.
A variety of molecules can be separated by capillary electrophoresis techniques. Sample types include simple organic molecules (charged or neutral), inorganic anions and cations, peptides, oligonucleotides, and DNA sequence fragments. Since the introduction of commercial instrumentation in 1988, the inherent capabilities of capillary electrophoresis and its various modes of operation have been widely demonstrated. Major advantages of capillary electrophoresis include high separation efficiency, small sample and reagent consumption, and low waste generation. The sample fragments in capillary electrophoresis are often analyzed by detecting light emission (e.g., from radiation induced fluorescence) or light absorption associated with the sample. The intensities of the emission are representative of the concentration, amount and/or size of the components of the sample.
Specifically, in capillary electrophoresis, separation is performed in small capillary tubes to reduce band broadening effects due to thermal convection and hence improve resolving power. By applying electrophoresis in a capillary column carrying a buffer solution, the sample size requirement is significantly smaller and the speed of separation and resolution can be increased multiple times compared to the slab gel-electrophoresis method. Only minute volumes of sample materials, typically less than 20 nanoliters, are required to be introduced into the separation capillary column.
It was mentioned in The Journal of Chromatography, 452, (1988) 615-622, that sample valves are the most suitable sampling method for capillary electrophoresis. The limitation of this method is the large sampling volume. A rotary injection valve has been used in capillary electrophoresis with a sampling volume of 350 nanoliters. The results have been reported in Anal. Chem. 59, (1987) 799. This volume is too large to be used for high-resolution separations. Later, an internal loop injection valve with an injection loop volume of ≧20 nL has become commercially available, but connecting capillaries to this valve is too much of a challenge and consequently it is not often used in capillary electrophoresis.
Current practical techniques for sample injection in capillary electrophoresis include electromigration and siphoning of sample from a container into one end of a separation column. For the siphoning injection technique, the sample reservoir is coupled to the inlet end of the capillary column and is raised above the buffer reservoir that is at the exit end of the capillary column for a fixed length of time. The electromigration injection technique is effected by applying an appropriate polarized electrical potential across the capillary column for a given duration while the entrance end of the capillary is in the sample reservoir. For both sample injection techniques the input end of the analysis capillary tube must be transferred from a sample reservoir to a buffer reservoir to perform separation. Thus, a mechanical manipulation is involved. It is also difficult to maintain consistency in injecting a fixed volume of sample by either of these techniques, as the sample volume injected are susceptible to changes in sample viscosity, temperature, etc., thereby resulting in relatively poor reproducibility in injected sample volumes between separation runs. Electromigration additionally suffers from electrophoretic mobility-based bias.
Electrophoresis based on microfabricated chips possesses many unique advantages over conventional capillary electrophoresis. One of them is the so-called “differential concentration” effect for separation of DNA sequencing fragments. For sequencing using conventional capillary gel electrophoresis, the signal intensity of separated fragment has an exponential profile against fragment size. That is, very high signal intensities for short fragments and very low for large fragments. Often, the readlength of DNA sequencing is limited by the low signal intensity rather than the resolution for the long fragments. This exponential profile also requires a wide dynamic range for detection.
Capillary electrophoresis on microchips is an emerging new technology that promises to lead the next revolution in chemical analysis. It has the potential to simultaneously assay hundreds of samples in minutes or less time. Microfluidic chips used in electrophoretic separations usually have dimensions from millimeters to decimeters. The largest electrophoretic separation chip so far has a substrate having dimensions of 50-cm×25-cm, which was disclosed in Micro Total Analysis Systems 2001, 16-18. These microfluidic platforms require only nanoliter or picoliters volumes of sample, in contrast to the microliter volumes required by other separation technologies. These samples may potentially be prepared on-chip for a complete integration of sample preparation and analysis functions. The rapid analysis combined with massively parallel analysis arrays could yield ultrahigh throughputs. These features make microchips an attractive technology for the next generation of capillary electrophoresis instrumentation.
These microchips are prepared using microfabrication techniques developed in the semiconductor industry. Capillary channels are fabricated in microchips using, for example, photolithography or micromolding techniques. Microchips have been demonstrated for separations of amino acids, DNA restriction fragments, PCR products, short oligonucleotides, and sequencing ladders.
For capillary electrophoresis separation on microchips, samples are usually introduced using either cross-channel or double-T sample injectors. The cross-channel injector has been disclosed in U.S. Pat. No. 6,001,229. As illustrated in FIG. 1a, the cross-channel injector is formed by orthogonally intersecting the separation-channel 6 with a cross-channel 5 and 5a connecting the sample reservoir 1 to an analyte waste reservoir 2. To load sample to the separation-channel 6, analytes are electrophoresed (e.g., by electrokinetic forces) from the sample reservoir 1 to the analyte waste reservoir 2, filling the whole cross-channel 5 including the intersection region 7. When an electric potential is applied to cathode reservoir 3 and anode reservoir 4 along the separation-channel 6 after analytes have been loaded into the intersection region 7, the analytes residing in the intersection region 7 are electrokinetically driven down the separation-channel 6 to perform electrophoretic separation.
In the sample loading process, as analytes migrate across the intersection region 7 analytes disperse orthogonally into the separation-channel 6 due to the electric field distortion and molecular diffusion. This degrades the resolving power and makes the separation irreproducible. To overcome this dispersion, selected voltages are applied to the cathode and anode reservoirs 3 and 4 such that buffer electrolytes, along with the dispersed analytes, are electrokinetically driven to the intersection region 7 and ultimately to the analyte waste reservoir 2. Therefore analyte dispersion is suppressed. This is called a “pinched” injection mode.
A double-T injector on microchips has been disclosed in U.S. Pat. No. 6,280,589. In a double-T injector (referring to FIG. 1b), the sample channel across the separation channel 6 is divided by the separation-channel into two segments 8 and 9 that are offset by a given distance along the separation channel 6. If the channel connecting the sample reservoir 1 and analyte waste reservoir 2 is still considered the “cross-channel”, the offset segment 10 is shared by the “cross-channel” and the separation-channel 6. Similar to the cross-channel injector in FIG. 1a, sample is loaded by electrophoresis, from the sample reservoir 1 to the analyte waste reservoir 2, filling the cross-channel segments 8 and 9 and the offset segment 10. As an electric potential is applied to cathode and anode reservoirs 3 and 4 across the separation-channel 6 (including the offset segment 10) after analytes have been loaded in the off-set segment 10, the analytes residing in the offset segment region 10 are electrokinetically driven down the separation-channel 6 to perform electrophoretic separation. Double-T injectors also suffer from dispersion of analytes into the separation channel. “Pinched” injection mode is usually used to suppress this problem, as discussed in Anal. Chem. 71 (1999) 566-573.
Precise control of the potentials on multiple electrodes in reservoirs 1, 2, 3 and 4 is critical to achieving desired and reproducible results for either cross-channel or double-T injectors, especially when a “pinched” injection mode is employed. These potentials are balanced and calibrated normally using a standard sample until reproducible results have been obtained. However, when samples of different ionic strength and viscosity are to be analyzed, that calibrated potential balance for the device is no longer applicable, and consequently giving rise to undesired and/or irreproducible results.
For sequencing separation on chips with a cross or a double-T sample injector, a uniform signal intensity profile is typically obtained. The mechanism has been illustrated in the Proc. Natl. Acad. Sci. U.S.A. 97 (2000) 5369-5374. During injection, sample is electrophoresed through the cross channel to the offset segment 10 (referring to FIG. 1b). This electrophoresis of DNA fragments provides differential enrichment of sequencing fragments. Little change in concentration will occur at the sample/gel interface for small DNA fragments and inorganic ions because their electrophoretic mobilities are similar in free solution and in sieving matrix. On the other hand, a considerable increase in the steady-state concentration will occur at the sample/gel interface for the large fragments because of their reduced mobility in the gel. These results in a concentration compensation for large fragments. Concentrations of large fragments are always lower than those of small fragments in a typical sequencing sample. A uniform intensity profile is therefore generated.
Another advantage of microchips is to use cross-channel 5 or 8 (referring to FIG. 1) to perform sample preseparation or cleanup. Taking DNA sequencing for example, when sample is electrophoresed through the cross channel 5 to the intersection region 7 or segment 8 to the offset segment 10, at an optimized injection time, the majority of the fragments have reached a steady-state concentration in the intersection region 7 or segment 10, while large template and enzyme molecules are still migrating in the cross channel 5 or 8. When voltages are switched to separation, only the fragments in the injector are injected into the separation channel during the separation, while DNA template and enzyme contaminants were removed from the separation channel. Removal of these large molecules has been reported essential to achieve high quality separations. In capillary gel electrophoresis (CGE), they are removed using offline membrane filters.
T-injectors may be used for sample introduction on microchips as well. In this scheme (referring to FIG. 1c), the analyte waste reservoir 2 and the channel 9 between the separation-channel 6 and analyte waste reservoir 2 in FIG. 1b are eliminated. Analytes are electrophoresed from the sample reservoir 1 through the half “cross-channel” 8 directly into the separation-channel 6. Since the other half of the “cross-channel” 9 is omitted, all analytes exit the half “cross-channel” 8 enter and build up in the separation-channel 6 as the sample loading process continues.
However, there are two major problems associated with the T-injector. The first problem is the augmented electrophoretic mobility-based bias. In a normal electrokinetic injection process of capillary electrophoresis, as the sample inlet end of a separation capillary is dipped directly into the sample solution and all analytes migrate into the separation capillary simultaneously, the electrophoretic mobility-based bias equals to the ratio of their electrophoretic mobilities. In this T-injection scheme, the sample reservoir 1 and the inlet end of the separation-channel 6 are separated by the half “cross-channel” 8. Fast-moving analytes have already migrated into the separation-channel 6 when slow-moving analytes are still migrating in the half “cross-channel” 8. As a result, fast-moving analytes are more preferentially introduced in T-injectors than in conventional capillary electrophoresis and therefore the electrophoretic mobility-based bias is augmented.
The second problem is the difficulty in precisely controlling a finite amount of analytes into the separation-channel 6. This problem is associated with the variation of length of the half “cross-channel” 8. In a microchip fabrication process, channels are photolithographically created and can be very precisely arranged. The reservoirs are holes drilled or physically attached and their positions and dimensions cannot be reproducibly and precisely produced. In T-injection schemes, the quantity of the analytes injected into the separation-channel 6 is normally controlled through timing of the applied electrical potential. Because analytes going to the separation-channel 6 have to pass through the half “cross-channel” 8, it is a significant challenge to attempt to control the timing so that only a given finite amount of analytes is allowed to migrate into the separation-channel 6. Variation of the length of this channel makes the problem even more challenging.
It is therefore desirable to develop a robust, reproducible and automated sample injection scheme for a microfabricated device, which would overcome the limitations in the prior art.